Human amyloid protein precursor homologue and Kunitz-type inhibitors

ABSTRACT

The present invention provides isolated DNA molecules comprising a DNA segment encoding a novel human amyloid protein precursor homologue and novel Kunitz-type inhibitors. Also provided are DNA constructs comprising a first DNA segment encoding a novel human amyloid protein precursor homologue or a novel Kunitz-type inhibitor wherein said first DNA segment is operably linked to additional DNA segments required for the expression for the first DNA segment, as well as host cells containing such DNA constructs and methods for producing proteins from the host cells.

CROSS REFERENCE

The present application is a continuation-in-part of Ser. No.07/985,692, filed Dec. 2, 1992, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Polymorphonuclear leukocytes (neutrophils or PMNs) and mononuclearphagocytes (monocytes) play an important part in tissue injury,infection, acute and chronic inflammation and wound healing. There issome indication that a number of chronic diseases are caused bypathological proteolysis due to overstimulation of the PMNs. Suchoverstimulation may be caused by, for instance, autoimmune response,chronic infection, tobacco smoke or other irritants, etc.

PMNs migrate from the blood to the site of inflammation and, followingappropriate stimulation, they release oxidant compounds (O₂., O₂ ⁻, H₂O₂ and HOCl ) as well as granules containing a variety of proteolyticenzymes. The secretory granules contain, inter alia, alkalinephosphatase; metalloproteinases such as gelatinase and collagenase; andserine proteases such as neutrophil elastase, cathepsin G andproteinase-3.

Latent metalloproteinases are released together with tissue inhibitor ofmetalloproteinase (TIMP). The activation mechanism has not been fullyelucidated, but it is likely that oxidation of thiol groups and/orproteolysis play a part in the process. Also, free metalloproteinaseactivity is dependent on inactivation of TIMP.

PMNs contain large quantities of serine proteases, and about 200 mg ofeach of the leukocyte proteases are released daily to deal with invasiveagents in the body. Acute inflammation leads to a many-fold increase inthe amount of enzyme released. In the azurophil granules ofpolymorphonuclear leukocytes, the serine proteases neutrophil elastase,cathepsin G and proteinase-3 are packed as active enzymes complexed withglucosaminoglycans. These complexes are inactive but dissociate onsecretion to release the active enzymes. Under normal conditions,proteolysis is kept at an acceptably low level by large amounts of theinhibitors α₁ -proteinase inhibitor (α₁ -PI), α₁ -chymotrypsin inhibitor(α₁ -ChI) and α₂ macroglobulin that are found in plasma. However, thePMNs are able to inactivate the inhibitors locally. Thus, α₁ -PI, whichis the most important inhibitor of neutrophil elastase, is sensitive tooxidation at the reactive center (Met-358) by oxygen metabolitesproduced by stimulated PMNs. This reduces the affinity of α₁ -PI forneutrophil elastase by approximately 2000 times.

After local neutralization of α₁ -PI, the neutrophil elastase is able todegrade a number of inhibitors of other proteolytic enzymes. Neutrophilelastase cleaves α₁ -ChI and thereby promotes cathepsin G activity. Italso cleaves TIMP, resulting in tissue degradation bymetalloproteinases. Furthermore, neutrophil elastase cleavesantithrombin III, heparin cofactor II, and tissue factor pathwayinhibitor (TFPI), which probably promotes clot formation. On the otherhand, the ability of neutrophil elastase to degrade coagulation factorsis assumed to have the opposite effect, such that the total effect ofneutrophil elastase is unclear. The effect of neutrophil elastase onfibrinolysis is less ambiguous. Fibrinolytic activity increases whenneutrophil elastase cleaves plasminogen activator inhibitor and α₂plasmin inhibitor. Furthermore, both of these inhibitors are oxidizedand inactivated by O₂ metabolites.

Protein inhibitors are classified into a series of families based onextensive sequence homologies among the family members and theconservation of intrachain disulfide bridges (for review, see Laskowskiand Kato, Ann. Rev. Biochem. 49: 593-626, 1980). Serine proteaseinhibitors of the Kunitz family are characterized by their homology withaprotinin (bovine pancreatic trypsin inhibitor). Aprotinin is known toinhibit various serine proteases including trypsin, chymotrypsin,plasmin and kallikrein. Kunitz-type inhibitor domains have been reportedin larger proteins such as the inter-α-trypsin inhibitors (Hochstrasseret al., Hoppe-Seylers Z. Physiol. Chem. 357: 1659-1661, 1969 andTschesche et al., Eur. J. Biochem. 16: 187-198, 1970) and the β-amyloidprotein precursor. The β-amyloid protein precursor (APP) contains anapproximately 40 amino acid fragment that forms the senile plaquesobserved in Alzheimer's patients, patients with Down's syndrome and inaged normal patients. The gene encoding APP yields three alternativelyspliced mRNAs, two of which have been demonstrated to encode Kunitz-typeinhibitor domains (see Ponte et al., Nature 331: 525-528, 1988; Tanzi etal., Nature 331: 528- 530, 1988 and Kitaguchi et al., Nature 331:530-532, 1988). In addition to the Kunitz-type inhibitor domain, eachprotein precursor contains a signal peptide, a cysteine-rich region, ahighly negatively charged region, a transmembrane domain and anintracellular domain (see Kitaguchi et al. ibid.).

Of the Kunitz-type inhibitors, aprotinin is used therapeutically in thetreatment of acute pancreatitis, various states of shock syndrome,hyperfibrinolytic hemorrhage and myocardial infarction (see, forexample, Trapnell et al., Brit. J. Surg. 61: 177, 1974; McMichan et al.,Circulatory shock 9: 107, 1982; Auer et al., Acta Neurochir. 49: 207,1979; Sher, Am. J. Obstet. Gynecol. 129: 164, 1977; and Schneider,Artzneim.-Forsch. 26: 1606, 1976). Administration of aprotinin in highdoses significantly reduces blood loss in connection with cardiacsurgery, including cardiopulmonary bypass operations (see, for example,Bidstrup et al., J. Thorac. Cardiovasc. Surg. 97: 364-372, 1989; vanOeveren et al., Ann. Thorac. Surg. 44: 640-645, 1987). It has previouslybeen demonstrated (Wenzel and Tschesche, Angew. Chem. Internat. Ed. 20:295, 1981) that certain aprotinin analogs, e.g. aprotinin (1-58, Val15),exhibit a relatively high selectivity for granulocyte elastase and aninhibitory effect on collagenase. Aprotinin (1-58, Ala15) has a weakeffect on elastase, while aprotinin (3-58, Arg15, Ala17, Ser42) exhibitsan excellent plasma kallikrein inhibitory effect (WO 89/10374).

However, when administered in vivo, aprotinin has been found to have anephrotoxic effect in rats, rabbits and dogs after repeated injectionsof relatively high doses (Bayer, Trasylol, Inhibitor of Proteinase;Glaser et al. in "Verhandlungen der Deutschen Gesellschaft fur InnereMedizin, 78. Kongress," Bergmann, Munich, 1972, pp. 1612-1614). Thenephrotoxicity (appearing, i.e., in the form of lesions) observed foraprotinin might be ascribed to the accumulation of aprotinin in theproximal tubulus cells of the kidneys as a result of the high positivenet charge of aprotinin, which causes it to be bound to the negativelycharged surfaces of the tubuli. This nephrotoxicity makes aprotinin lesssuitable for clinical purposes, particularly in those uses requiringadministration of large doses of the inhibitor (such as cardiopulmonarybypass operations). Furthermore, aprotinin is a bovine protein, whichmay induce an immune response upon administration to humans.

It is therefore an object of the present invention to provide novelhuman protease inhibitors of the Kunitz family of inhibitors withsimilar inhibitor profiles for use in the treatment of acutepancreatitis, various states of shock syndrome, hyperfibrinolytichemorrhage and myocardial infarction. It is further an object of thepresent invention to provide novel amyloid protein precursor homologuesfor use in studying the relative levels of the precursor in patientsexhibiting Alzheimer's disease and to identify patients with mutationsin the protein precursor.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides novel isolated DNAmolecules encoding human amyloid protein precursor homologues and humanKunitz-type inhibitors. Within one embodiment of the invention, the DNAmolecule comprises a DNA segment encoding a human amyloid proteinprecursor homologue, wherein the DNA segment comprises the sequence ofnucleotides of SEQ ID NO:12 from nucleotide 73 to nucleotide 2361.Within another embodiment, the DNA segment encodes a human amyloidprotein precursor homologue comprising the amino acid sequence of SEQ IDNO:13 from methionine, amino acid number 1, to isoleucine, amino acidnumber 763. Also provided are DNA constructs comprising a first DNAsegment encoding a human amyloid protein precursor homologue operablylinked to additional DNA segments necessary for the expression of thefirst DNA segment, host cells containing such DNA constructs, as well asmethods for producing a human amyloid protein precursor homologuecomprising the step of culturing a host cell under conditions promotingthe expression of a DNA segment encoding a human amyloid precursorprotein homologue.

Within another aspect of the invention, DNA molecules are provided whichcomprise a DNA segment encoding a human Kunitz-type inhibitor, whereinthe DNA segment comprises the sequence of nucleotides of SEQ ID NO:1from nucleotide 171 to nucleotide 331 or the sequence of nucleotides ofSEQ ID NO:1 from nucleotide 159 to nucleotide 331. Within anotherembodiment, the DNA segment encodes a human Kunitz-type inhibitorcomprising the amino acid sequence of SEQ ID NO:2 from alanine, aminoacid 56 to alanine, amino acid number 110; the amino acid sequence ofSEQ ID NO:2 from aspartic acid, amino acid number 53 to alanine, aminoacid number 110; or the amino acid sequence of SEQ ID NO:2 from valine,amino acid number 57 to alanine, amino acid number 110 and which furthercontains a glutamic acid residue on the amino terminus. Also providedare DNA constructs comprising a first DNA segment encoding a humanKunitz-type inhibitor operably linked to additional DNA segmentsnecessary for the expression of the first DNA segment, host cellscontaining such DNA constructs, as well as methods for producing a humanKunitz-type inhibitor comprising the step of culturing a host cell underconditions promoting the expression of a DNA segment encoding a humanKunitz-type inhibitor.

Within another aspect of the invention, isolated amyloid protenprecursor homologues and Kunitz-type inhibitors are provided. Within oneembodiment, an isolated amyloid protein precursor homologue comprisesthe amino acid sequence of SEQ ID NO:13 from methionine, amino acidnumber 1, to isoleucine, amino acid number 763. Within anotherembodiment, an isolated human Kunitz-type inhibitor comprises the aminoacid sequence of SEQ ID NO:2 from alanine, amino acid 56 to alanine,amino acid number 110; the amino acid sequence of SEQ ID NO:2 fromaspartic acid, amino acid number 53 to alanine, amino acid number 110;or the amino acid sequence of SEQ ID NO:2 from valine, amino acid number57 to alanine, amino acid number 110 and which further contains aglutamic acid residue on the amino terminus.

Within another aspect of the invention, isolated antibodies are providedwith specifically bind to a human amyloid protein precursor or to ahuman Kunitz-type inhibitor. Within one embodiment, the antibody is amonoclonal antibody.

Within yet another aspect of the invention, a pharmaceutical compositionis provided which comprises a human Kunitz-type inhibitor comprising theamino acid sequence of SEQ ID NO:2 from alanine, amino acid 56 toalanine, amino acid number 110; the amino acid sequence of SEQ ID NO:2from aspartic acid, amino acid number 53 to alanine, amino acid number110; or the amino acid sequence of SEQ ID NO:2 from valine, amino acidnumber 57 to alanine, amino acid number 110 and which further contains aglutamic acid residue on the amino terminus.

Within another aspect of the invention, probes of at least 12nucleotides are provided, wherein the probes are capable of hybridizingwith nucleic acids encoding a human amyloid protein precursor homologuecomprising the nucleotide sequence of SEQ ID NO:12, nucleotide variantsof SEQ ID NO:12, or DNA segments encoding DNA sequences complementary toSEQ ID NO:12 or its variants. Within yet another aspect of theinvention, probes of at least 12 nucleotides are provided wherein theprobes are capable of hybridizing with nucleic acids encoding aKunitz-type inhibitor domain comprising the nucleotide sequence of SEQID NO:1, nucleotide variants of SEQ ID NO:1, or DNA segments encodingDNA sequences complementary to SEQ ID NO:1 or its variants.

These and other aspects will become evident upon reference to thefollowing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel human amyloid protein precursorhomologue containing a protease inhibitor domain that shares homologywith the Kunitz family of protease inhibitors and a novel placentalkunitz-type inhibitor. One advantage of the inhibitors of the presentinvention is that, contrary to aprotinin, the inhibitors of the presentinvention are human proteins, so that undesired immunological reactionson administration to humans are significantly reduced. The Kunitz-typeinhibitor of the amyloid protein precursor homologue has the advantagethat it has a negative net charge as opposed to aprotinin, which maythereby reduce the risk of kidney damage on administration of largedoses of the inhibitor. The amyloid protein precursor homologues of thepresent invention and the DNA sequences encoding such proteins providethe advantage of allowing the expression of such proteins in patientswith Alzheimer's disease to be compared with the expression of suchprotein in normal patients.

Features of the present invention include isolated DNA moleculesencoding novel human Kunitz-type inhibitors. Another feature of thepresent invention is an isolated DNA molecule encoding a human amyloidprotein precursor homologue. Such isolated molecules are those that areseparated from their natural environment and include cDNA and genomicclones. Isolated DNA molecules of the present invention are providedfree of other genes with which they are naturally associated and mayinclude naturally occurring 5' and 3' untranslated sequences thatrepresent regulatory regions such as promoters and terminators. Theidentification of regulatory regions within the naturally occurring 5'and 3' untranslated regions will be evident to one of ordinary skill inthe art (for review, see Dynan and Tijan, Nature 316: 774-778, 1985;Birnstiel et al., Cell 41: 349-359, 1985; Proudfoot, Trends in Biochem.Sci. 14: 105-110, 1989; and Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; whichare incorporated herein by reference).

The isolated DNA molecules of the present invention are useful inproducing recombinant human Kunitz-type inhibitors and recombinant humanamyloid protein precursor homologues. Thus, the present inventionprovides the advantage that human Kunitz-type inhibitors and amyloidprotein precursor homologues are produced in high quantities that may bereadily purified using methods known in the art (see generally, Scopes,Protein Purification, Springer-Verlag, N.Y., 1982). Alternatively, theproteins of the present invention can be synthesized following anysuitable method, such as by exclusively solid-phase techniques (i.e.,the method of Barany and Merrifield (in The Peptides. Analysis,Synthesis, Biology Vol. 2, Gross and Meienhofer, eds, Academic Press,NY, pp. 1-284, 1980)), by partial solid-phase techniques, by fragmentcondensation or by classical solution addition.

Kunitz-type inhibitor activity can be measured using the methodessentially described by Norris et al. (Biol. Chem. Hoppe-Seyler 371:37-42, 1990). Briefly, various fixed concentrations of the Kunitz-typeinhibitor are incubated in the presence of serine proteases at theconcentrations listed in Table 2 in 100 mM NaCl, 50 mM Tris HCl, 0.01%TWEEN80 (Polyoxyethylenesorbitan monoleate) (pH 7.4) at 25° C. After a30 minute incubation, the residual enzymatic activity is measured by thedegradation of a solution of the appropriate substrate as listed inTable 2 in assay buffer. The samples are incubated for 30 minutes afterwhich the absorbance of each sample is measured at 405 nm. An inhibitionof enzyme activity is measured as a decrease in absorbance at 405 nm orfluoresence Em at 460 nm. From the results, the apparent inhibitionconstant K_(i) is calculated.

The Kunitz-type inhibitors of the present invention may be used in thedisclosed methods for the treatment of, inter alia, acute pancreatitis,various states of shock syndrome, hyperfibrinolytic hemorrhage andmyocardial infarction. The amyloid protein precursor homologues of thepresent invention may be used, inter alia, to generate antibodies foruse in demonstrating tissue distribution of the precursor or for use inpurifying such proteins.

Thus, an additional feature of the present invention is an isolatedhuman Kunitz-type inhibitor or an isolated human amyloid proteinprecursor homologue. Isolated proteins and peptides of the presentinvention are proteins of at least about 50% homogeneity, morepreferably of 70% to 80% homogeneity with a protein preparation of 95%to 99% or more homogeneity most preferred, particularly forpharmaceutical uses.

The present invention relates to a novel human amyloid protein precursorhomologue containing a human Kunitz-type inhibitor comprising the aminoacid sequence shown in Sequence ID NO:13 and/or encoded by a DNAsequence comprising the nucleotide sequence of SEQ ID NO:12. TheKunitz-type inhibitors of the present invention are up to 80 aminoacids, preferably between 50 and 60 amino acids, most preferably between53 and 57 amino acids in length and comprise the amino acid sequenceshown SEQ ID NO:2 from valine, amino acid number 57 through alanine,amino acid number 110. However, as will be evident to one skilled in theart, amino-terminal and/or carboxy-terminal extensions of theKunitz-type inhibitor may be prepared either synthetically or usingrecombinant DNA techniques and tested for inhibitor activity.

The DNA sequences encoding the proteins of the present invention wereunexpectedly identified during screening for a cDNA corresponding to thegenomic clone of an unrelated Kunitz-type inhibitor using an antisenseoligonucleotide probe corresponding to a portion of the inhibitor codingsequence. Analysis of the cDNA clones revealed that the clones encoded aunique, previously unknown amyloid protein precursor homologuecontaining a Kunitz-type inhibitor domain and a novel placentalKunitz-type inhibitor. As used herein, the proteins of the presentinvention may be encoded by DNA sequences that are substantially similarto the DNA sequence disclosed herein. As used within the context of thepresent invention, "substantially similar" DNA sequences encompassallelic variants and genetically engineered or synthetic variants of theamyloid protein precursor homologue gene, Kunitz-type inhibitor domainof the amyloid protein precursor homologue gene, or the novel placentalKunitz-type inhibitor gene that contain conservative amino acidsubstitutions and/or minor additions, substitutions or deletions ofamino acids. DNA sequence variants also encompass degeneracies in theDNA code wherein host-preferred codons are substituted for the analogouscodons in the human sequence. In addition, substantially similar DNAsequences are those that are capable of hybridizing to the DNA sequencesof the present invention under high or low stringency (see Sambrook etal., ibid.) and those sequences that are degenerate as a result of thegenetic code to the amino acid sequences of the present invention.Genetically engineered variants may be obtained by usingoligonucleotide-directed site-specific mutagenesis, by use ofrestriction endonuclease digestion and adapter ligation, or othermethods well established in the literature (see for example, Sambrook etal., ibid. and Smith et al., Genetic Engineering: Principles andMethods, Plenum Press, 1981; which are incorporated herein byreference).

DNA sequences of the present invention may be isolated using standardcloning methods such as those described by Maniatis et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982; which isincorporated herein by reference), Sambrook et al. (Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; whichis incorporated herein by reference) or Mullis (U.S. Pat. No. 4,683,195;incorporated herein by reference). Alternatively, the coding sequencesof the present invention may be synthesized using standard techniquesthat are well known in the art, such as by synthesis on an automated DNAsynthesizer. As will be discussed in more detail below, a novel humanamyloid protein precursor homologue containing a Kunitz-type inhibitordomain was identified as a 3.5 kb cDNA insert and comprises the DNAsequence of SEQ ID NO:12. In a preferred embodiment, DNA sequencesencoding Kunitz-type inhibitors are obtained by PCR amplification usingprimers designed from SEQ ID NO:1 or its complement.

DNA molecules of the present invention or portions thereof may be used,for example, to directly detect amyloid protein precursor homologuesequences in cells. The DNA molecules of the present invention may alsobe used to detect kunitz-inhibitor sequences in cells. Such DNAmolecules are generally synthetic oligonucleotides, but may be generatedfrom cloned cDNA or genomic sequences and will generally comprise morethan 12 nucleotides, more often from about 14 nucleotides to about 25 ormore nucleotides, sometimes 40 to 60 nucleotides, and in some instancesa substantial portion or even the entire amyloid protein precursorhomologue gene or cDNA. The synthetic oligonucleotides of the presentinvention share at least about 85% identity, preferably at least 90%,and more preferably at least about 95% or more identity with acorresponding DNA sequence of the human amyloid protein precursorhomologue of SEQ ID NO:12, SEQ ID NO:1 or the complement of eithersequence. For use a probes, the molecules may be labeled to provide adetectable signal, such as with an enzyme, biotin, a radionuclide,fluorophore, chemiluminescer, paramagnetic particle, etc. according tomethods known in the art. Probes of the present invention may be useddiagnostic methods to detect cellular metabolic disorders such asthrombolic disorders.

DNA molecules used within the present invention may be labeled and usedin a hybridization procedure similar to the Southern or dot blot. Aswill be understood by those skilled in the art, conditions that allowthe DNA molecules of the present invention to hybridize to amyloidprotein precursor homologue sequences or amyloid protein precursorhomologue-like sequences may be determined by methods well known in theart and reviewed, for example, by Sambrook et al. (Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; whichis incorporated herein by reference). Those skilled in the art will becapable of varying hybridization conditions (i.e. stringency ofhybridization) of the DNA molecules as appropriate for use in thevarious procedures by methods well known in the literature (see, forexample, Sambrook et al., ibid., pages 11.45-11.53). The higher thestringency of hybridization, the lower the number of mismatchedsequences are detected. Alternatively, lower stringency will allowrelated sequences to be identified.

Alternatively, human amyloid protein precursor homologue sequencevariants may be identified using DNA molecules of the present inventionand, for example, the polymerase chain reaction (PCR) (disclosed bySaiki et al., Science 239: 487, 1987; Mullis et al., U.S. Pat. No.4,686,195; and Mullis, U.S. Pat. No. 4,683,202) to amplify DNAsequences, which are subsequently detected by their characteristic sizeon agarose gels or which may be sequenced to detect sequenceabnormalities.

Amyloid protein precursor homologue and Kunitz-type inhibitor sequencesof the present invention may be inserted into DNA constructs. As usedwithin the context of the present invention, a DNA constructs also knownas an expression vector, is understood to refer to a DNA molecule, or aclone of such a molecule, either single- or double-stranded, which hasbeen modified through human intervention to contain segments of DNAcombined and juxtaposed in a manner that would not otherwise exist innature. DNA constructs of the present invention comprise a first DNAsegment encoding an amyloid protein precursor homologue or a Kunitz-typeinhibitor operably linked to additional DNA segments required for theexpression of the first DNA segment. Within the context of the presentinvention, additional DNA segments will generally include promoters andtranscription terminators, and may further include enhancers and otherelements. One or more selectable markers may also be included.

In one embodiment the DNA sequence encodes a Kunitz-type inhibitorcomprising the amino acid sequence of SEQ ID NO:2 from alanine, aminoacid number 56 through alanine, amino acid number 110. In anotherembodiment, the first DNA sequence encodes a Kunitz-type inhibitorcomprising the amino acid sequence of SEQ ID NO:2 from aspartic acid,amino acid 53 to alanine, amino acid 110. In yet another embodiment, thefirst DNA sequence encodes a Kunitz-type inhibitor comprising the aminoacid sequence of SEQ ID NO:2 from valine, amino acid number 57 throughalanine, amino acid number 110 and which further contains a codon forglutamic acid immediately upstream of the codon for valine, amino acidnumber 57.

DNA constructs may also contain DNA segments necessary to direct thesecretion of a polypeptide or protein of interest. Such DNA segments mayinclude at least one secretory signal sequence. Secretory signalsequences, also called leader sequences, prepro sequences and/or presequences, are amino acid sequences that act to direct the secretion ofmature polypeptides or proteins from a cell. Such sequences arecharacterized by a core of hydrophobic amino acids and are typically(but not exclusively) found at the amino termini of newly synthesizedproteins. Very often the secretory peptide is cleaved from the matureprotein during secretion. Such secretory peptides contain processingsites that allow cleavage of the secretory peptides from the matureproteins as the pass through the secretory pathway. A preferredprocessing site is a dibasic cleavage site, such as that recognized bythe Saccharomyces cerevisiae KEX2 gene. A particularly preferredprocessing site is a Lys-Arg processing site. Processing sites may beencoded within the secretory peptide or may be added to the peptide by,for example, in vitro mutagenesis.

Preferred secretory signals include the α factor signal sequence (preprosequence: Kurjan and Herskowitz, Cell 30: 933-943, 1982; Kurjan et al.,U.S. Pat. No. 4,546,082; Brake, EP 116,201), the PHO5 signal sequence(Beck et al., WO 86/00637), the BAR1 secretory signal sequence (MacKayet al., U.S. Pat. No. 4,613,572; MacKay, WO 87/002670), and the SUC2signal sequence (Carlsen et al., Molecular and Cellular Biology 3:439-447, 1983). Alternately, a secretory signal sequence may besynthesized according to the rules established, for example, by vonHeinje (European Journal of Biochemistry 133: 17-21, 1983; Journal ofMolecular Biology 184: 99-105, 1985; Nucleic Acids Research 14:4683-4690, 1986). A particularly preferred signal sequence is thesynthetic signal LaC212 spx (1-47)--ERLE described in WO 90/10075, whichis incorporated by reference herein in its entirety.

Secretory signal sequences may be used singly or may be combined. Forexample, a first secretory signal sequence may be used in combinationwith a sequence encoding the third domain of barrier (described in U.S.Pat. No. 5,037,243, which is incorporated by reference herein in itsentirety). The third domain of barrier may be positioned in properreading frame 3' of the DNA segment of interest or 5' to the DNA segmentand in proper reading frame with both the secretory signal sequence anda DNA segment of interest.

The choice of suitable promoters, terminators and secretory signals iswell within the level of ordinary skill in the art. Methods forexpressing cloned genes in Saccharomyces cerevisiae are generally knownin the art (see, "Gene Expression Technology, " Methods in Enzymology,Vol. 185, Goeddel (ed.), Academic Press, San Diego, Calif., 1990 and"Guide to Yeast Genetics and Molecular Biology," Methods in Enzymology,Guthrie and Fink (eds.), Academic Press, San Diego, Calif., 1991; whichare incorporated herein by reference). Proteins of the present inventioncan also be expressed in filamentous fungi, for example, strains of thefungi Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349, which isincorporated herein by reference). Expression of cloned genes incultured mammalian cells and in E. coli, for example, is discussed indetail in Sambrook et al. (Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., 1989; which is incorporatedherein by reference). As would be evident to one skilled in the art, onecould express the proteins of the instant invention in other host cellssuch as avian, insect and plant cells using regulatory sequences,vectors and methods well established in the literature.

In yeast, suitable yeast vectors for use in the present inventioninclude YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039,1978), YEp13 (Broach et al., Gene 8: 121-133, 1979), POT vectors(Kawasaki et al, U.S. Patent No. 4,931,373, which is incorporated byreference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978)and derivatives thereof. Preferred promoters for use in yeast includepromoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or alcoholdehydrogenase genes (Young et al., in Genetic Engineering ofMicroorganisms for Chemicals, Hollaender et al., (eds.), p. 355, Plenum,N.Y., 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983). In this regard,particularly preferred promoters are the TPI1 promoter (Kawasaki, U.S.Pat. No. 4,599,311, 1986) and the ADH2-4^(c) promoter (Russell et al.,Nature 304: 652-654, 1983; Irani and Kilgore, U.S. patent applicationSer. No. 07/784,653, CA 1,304,020 and EP 284 044, which are incorporatedherein by reference). The expression units may also include atranscriptional terminator. A preferred transcriptional terminator isthe TPI1 terminator (Alber and Kawasaki, ibid.).

Host cells containing DNA constructs of the present invention are thencultured to produce amyloid protein precursor homologues or Kunitz-typeinhibitors. The cells are cultured according to standard methods in aculture medium containing nutrients required for growth of theparticular host cells. A variety of suitable media are known in the artand generally include a carbon source, a nitrogen source, essentialamino acids, vitamins, minerals and growth factors. The growth mediumwill generally select for cells containing the DNA construct by, forexample, drug selection, complementation of glycolytic gene defect orcomplementation of a deficiency in an essential nutrient by a selectablemarker on the DNA construct or co-transfected with the DNA construct.

Yeast cells, for example, are preferably cultured in a chemicallydefined medium, comprising a non-amino acid nitrogen source, inorganicsalts, vitamins and essential amino acid supplements. The pH of themedium is preferably maintained at a pH greater than 2 and less than 8,preferably at pH 6.5. Methods for maintaining a stable pH includebuffering and constant pH control, preferably through the addition ofsodium hydroxide. Preferred buffering agents include succinic acid and.Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Yeast cells having adefect in a gene required for asparagine-linked glycosylation arepreferably grown in a medium containing an osmotic stabilizer. Apreferred osmotic stabilizer is sorbitol supplemented into the medium ata concentration between 0.1M and 1.5M, preferably at 0.5M or 1.0M.Cultured mammalian cells are generally cultured in commerciallyavailable serum-containing or serum-free media. Selection of a mediumappropriate for the particular host cell used is within the level ofordinary skill in the art.

Within one embodiment of the invention, the proteins of the presentinvention are expressed in mammalian cells. Methods for introducingexogenous DNA into mammalian host cells include calciumphosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Vander Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.1:841-845, 1982) and DEAE-dextran mediated transfection (Ausubel et al.,eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc.,N.Y., 1987), which are incorporated herein by reference. The productionof recombinant proteins in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134, which are incorporated herein byreference. Preferred cultured mammalian cells include the COS-1 (ATCCNo. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK570 (ATCC No. CRL 10314) and 293 (ATCC No. CRL 1573; Graham et al., J.Gen. Virol. 36:59-72, 1977) cell lines. Additional suitable cell linesare known in the art and available from public depositories such as theAmerican Type Culture Collection, Rockville, Md.

The recombinant amyloid protein precursor homologues or Kunitz-typeinhibitors expressed using the methods described herein are isolated andpurified by conventional procedures, including separating the cells fromthe medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulfate, purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography or affinitychromatography, or the like. Methods of protein purification are knownin the art (see generally, Scopes, R., Protein Purification,Springer-Verlag, N.Y. (1982), which is incorporated herein by reference)and may be applied to the purification of the recombinant proteins ofthe present invention.

Preferably, the Kunitz-type inhibitors of the present invention arepurified using the method essentially described by Norris et al. (Biol.Chem. Hoppe-Seyler 371: 37-42, 1990, which is incorporated by referenceherein in its entirety). Briefly, selected yeast transformants are grownin 10 liters of YEPD for approximately 40 hours at 30° C. until an OD₆₀₀of approximately 25 has been reached. The culture is centrifuged, andthe supernatant is decanted. A 300 ml-1000 ml aliquot of supernatant isadjusted to pH 2.3 and applied to a column holding 8 ml of S-Sepharaose(Pharmacia-LKB Biotechnology AS, Alleroed, Denmark) or the like that hasbeen previously equilibrated with 20 mM Bicine, pH 8.7 (Sigma ChemicalCo., St. Louis, Mo.). After the column has been extensively washed with20 mM Bicine, pH 8.7, the Kunitz-type inhibitor is eluted with 30 ml of20 mM Bicine, pH 8.7 containing 1M NaCl. The eluted material is desaltedby application to a Sephadex G-25 column (a beaded dextran matrix,Pharmacia-LKB Biotechnology AS, Alleroed, Denmark; 2.5×30 cm) or thelike that has been equilibrated with 20 mM NH₄ HCO₃, pH 7.8. TheKunitz-type inhibitor is eluted with 20 mM NH₄ HCO₃, pH 7.8.

The Kunitz-type inhibitor is further purified and concentrated bychromatography on a Mono S column (Pharmacia-LKB Biotechnology AS,Alleroed, Denmark; 0.5×5 cm) or the like equilibrated with 20 mM Bicine,pH 8.7. After washing with the equilibration buffer at 2 ml/min for 10minutes, gradient elution of the Kunitz-type inhibitor is carried outover twelve minutes at 1 ml/min from 0-0.6M NaCl in the equilibrationbuffer. Peak samples are pooled, and the Kunitz-type inhibitor ispurified using reverse phase HPLC on a Vydac 214TP510 column (Mikro-lab,Aarhus, Denmark; 1.0×25 cm) or the like with a gradient elution at 4ml/min from 5% A (0.1% trifluoroacetic acid (TFA) in water) to 45% B(0.7% TFA in acetonitrile) in 20 minutes. The purified product inlyophilized in water, and inhibitor activity is measured.

The present invention also relates to a pharmaceutical compositioncomprising a Kunitz-type inhibitor of the present invention togetherwith a pharmaceutically acceptable carrier or vehicle. In thecomposition of the invention, the Kunitz-type inhibitor may beformulated by any of the established methods of formulatingpharmaceutical compositions, e.g. as described in Remington'sPharmaceutical Sciences, 1985. The composition may typically be in aform suited for systemic injection or infusion and may, as such, beformulated with sterile water or an isotonic saline or glucose solution.

Kunitz-type inhibitors of the present invention are thereforecontemplated to be advantageous to use for the therapeutic applicationssuggested for native aprotinin or aprotinin analogs with other inhibitorprofiles, in particular those which necessitate the use of largeaprotinin doses. Therapeutic applications for which the use of theKunitz-type inhibitor of the invention is indicated as a result of itsability to inhibit human serine proteases, e.g. trypsin, plasmin,kallikrein, elastase, cathepsin G and proteinase-3, include (but are notlimited to) acute pancreatitis, inflammation, thrombocytopenia,preservation of platelet function, organ preservation, wound healing,shock (and conditions involving hyperfibrinolytic hemorrhage, emphysema,rheumatoid arthritis, adult respiratory distress syndrome, chronicinflammatory bowel disease and psoriasis, in other words diseasespresumed to be caused by pathological proteolysis by elastase, cathepsinG and proteinase-3 released from triggered PMNs.

Furthermore, the present invention relates to the use of the Kunitz-typeinhibitors as described above for the preparation of a medicament forthe prevention or therapy of diseases or conditions associated withpathological proteolysis by proteases released from overstimulated PMNs.As indicated above, it may be an advantage to administer heparinconcurrently with the Kunitz-type inhibitors of the present invention.

Apart from the pharmaceutical use indicated above, the Kunitz-typeinhibitors as specified above may be used to isolate useful naturalsubstances, e.g. proteases or receptors from human material, which binddirectly or indirectly to the Kunitz-type inhibitor, for instance byscreening assays or by affinity chromatography.

Within one aspect of the present invention, amyloid protein precursorhomologues and Kunitz-type inhibitors, including derivatives thereof, aswell as portions or fragments of these proteins, are utilized to prepareantibodies which specifically bind to the amyloid protein precursorhomologues and Kunitz-type inhibitors. As used herein, the term"antibodies" includes polyclonal antibodies, monoclonal antibodies,antigen-binding fragments thereof such as F(ab')₂ and Fab fragments, aswell as recombinantly produced binding partners. These binding partnersincorporate the variable regions from a gene which encodes aspecifically binding monoclonal antibody. Antibodies are defined to bespecifically binding if they bind to the amyloid protein precursorhomologue or Kunitz-type inhibitor with a K_(a) of greater than or equalto 10⁷ /M. The affinity of a monoclonal antibody or binding partner maybe readily determined by one of ordinary skill in the art (see,Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

Methods for preparing polyclonal and monoclonal antibodies have beenwell described in the literature (see for example, Sambrook et al.,ibid.; Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:Techniques and Applications, CRE Press, Inc., Boca Raton, Fla., 1982).As would be evident to one of ordinary skill in the art, polyclonalantibodies may be generated from a variety of warm-blooded animals suchas horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats.The immunogenicity of an amyloid protein precursor homologue orKunitz-type inhibitor may be increased through the use of an adjuvantsuch Freund's complete or incomplete adjuvant. A variety of assays knownto those skilled in the art may be utilized to detect antibodies whichspecifically bind to an amyloid protein precursor homologue orKunitz-type inhibitor. Exemplary assays are described in detail inAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold SpringHarbor Laboratory Press, 1988. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radio-immunoassays,radio-immunoprecipitations, enzyme-linked immuno-sorbent assays, dotblot assays, inhibition or competition assays, and sandwich assays.

Additional techniques for the preparation of monoclonal antibodies mayutilized to construct and express recombinant monoclonal antibodies.Briefly, mRNA is isolated from a B cell population and utilized tocreate heavy and light chain immunoglobulin cDNA expression libraries ina suitable vector such as the λ IMMUNOZAP(H) and λIMMUNOZAP(L) vectors,which may be obtained from Stratocyte (La Jolla, Calif.). These vectorsare then screened individually or are co-expressed to form Fab fragmentsor antibodies (Huse et al., Science 246: 1275-1281, 1989; Sastry et al.,Proc. Natl. Acad. Sci. USA 86: 5728-5732, 1989). Positive plaques aresubsequently converted to a non-lytic plasmid which allows high levelexpression of monoclonal antibody fragments in E. coli.

Binding partners such as those described above may also be constructedutilizing recombinant DNA techniques to incorporate the variable regionsof a gene which encodes a specifically binding antibody. Theconstruction of these proteins may be readily accomplished by one ofordinary skill in the art (see for example, Larrick et al.,Biotechnology 7: 934-938, 1989; Reichmann et al., Nature 322: 323-327,1988 and Roberts et al. Nature 328: 731-734, 1987). Once suitableantibodies or binding partners have been obtained, they may be isolatedor purified by many techniques well described in the literature (see forexample, Antibodies: A Laboratory Manual, ibid.). Suitable techniquesinclude protein or peptide affinity columns, HPLC or RP-HPLC,purification on protein A or protein G columns or any combination ofthese techniques. Within the context of the present invention, the term"isolated" as used to define antibodies or binding partners means"substantially free of other blood components."

Antibodies and binding partners of the present invention may be used ina variety of ways. The tissue distribution of amyloid protein precursorhomologues, for example, may be determined by incubating tissue sliceswith a labeled monoclonal antibody which specifically binds to theamyloid protein precursor homologue, followed by detection of thepresence of the bound antibody. Labels suitable for use within thepresent invention are well known in the art and include, among others,fluorescein, isothiocyanate, phycoerythrin, horseradish peroxidase, andcolloidal gold. The antibodies of the present invention may also be usedfor the purification of amyloid protein precursor homologues andKunitz-type inhibitors of the present invention. The coupling ofantibodies to solid supports and their use in purification of proteinsis well known in the literature (see for example, Methods in MolecularBiology, Vol. 1, Walker (Ed.), Humana Press, New Jersey, 1984, which isincorporated by reference herein in its entirety).

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLES

Restriction endonucleases and other DNA modification enzymes (e.g., T4polynucleotide kinase, calf alkaline phosphatase, DNA polymerase I(Klenow fragment), T4 polynucleotide ligase) were obtained from GIBCOBRL Life Technologies, Inc (GIBCO BRL) and New England Biolabs and wereused as directed by the manufacturer, unless otherwise noted.

Oligonucleotides were synthesized on an Applied Biosystems Model 380ADNA synthesizer and purified by polyacrylamide gel electrophoresis ondenaturing gels. E. coli cells were transformed as described by Maniatiset al. (Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, 1982) or Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2 ed., Cold Spring Harbor, N.Y., 1989). Radiolabeled probes andhybridization solutions were prepared essentially as described bySambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed., ColdSpring Harbor, N.Y., 1989; which is incorporated by reference herein inits entirety).

EXAMPLE 1 Cloning of An Amyloid Precursor Protein cDNA

Poly(A)⁺ RNAs from a variety of human tissue sources were screened usingan antisense 30-mer oligonucleotide (ZC4792; SEQ ID NO:5). A blot ofhuman poly(A)⁺ RNA from heart, brain, placenta, liver, lung, skeletalmuscle, kidney and pancreas (HUMAN MTN BLOT) was obtained from ClontechLaboratories, Inc. (Palo Alto, Calif.). The blot was prehybridized in aprehybridization solution containing 5× SSPE (Table 1), 2× Denhardt's(Table 1), 0.5% sodium dodecyl sulfate (SDS), and 100 μg/ml sonicatedsalmon sperm DNA for four hours at 55° C. After prehybridization, theprehybridization solution was removed and replaced with prehybridizationsolution containing 4.7×10⁶ cpm/ml of ³² P-labeled ZC4792 (SEQ ID NO:5).After an overnight incubation at 55° C. the hybridization solution wasremoved, and the blot was washed once in 2× SSC (Table 1), 0.05% SDS atroom temperature for 20 minutes followed by a wash in 2× SSC (Table 1),0.1% SDS for 20 minutes at 55° C. The blot was exposed to film for twoand a half hours. The resulting autoradiograph showed a number of bandsin most of the lanes, indicating the presence of related sequences inmost of the tissues represented in the blot. The blot was washed at ahigher stringency in 2× SSC (Table 1) at 60°-65° C. for 30 minutes,after which the blot was exposed to film overnight. The secondautoradiograph showed the presence of a 1.6 kb band for placenta andliver and an apparently smaller band of approximately 1.2 kb in thepancreas.

                  TABLE 1                                                         ______________________________________                                        20× SSPE                                                                175.3 g NaCl                                                                  27.6 g NaH.sub.2 PO.sub.4.H.sub.2 O                                           7.4 g EDTA                                                                    Dissolve the solids in 800 ml of distilled                                    water. Adjust the pH to 7.4 with NaOH                                         (approximately 6.5 ml of a 10 N solution).                                    Adjust the volume to 1 liter with distilled                                   water. Sterilize by autoclaving.                                              50× Denhardt's                                                          5 g Ficoll                                                                    5 g polyvinylpyrrolidone                                                      5 g bovine serum albumin (Fraction V)                                         Dissolve the solids into a final volume of 500                                ml. Filter the solution to sterilize and store                                at -20° C.                                                             20× SSC                                                                 175.3 g NaCl                                                                  88.2 g sodium citrate                                                         Dissolve the solids in 800 ml of distilled                                    water. Adjust the pH to 7.0 by a drop-wise                                    addition of 10 N NaOH. Adjust the volume to 1                                 liter with distilled water. Sterilize by                                      autoclaving.                                                                  Prehybridization Solution #1                                                  5× SSPE                                                                 5× Denhardt's                                                           0.5% SDS                                                                      100 μg/ml sheared salmon sperm DNA                                         Prehybridization Solution #2                                                  5× SSC                                                                  5× Denhardt's                                                           0.1% SDS                                                                      100 μg/ml sheared salmon sperm DNA                                         Growth Medium                                                                 Dulbecco's Modified Eagle's Medium (DMEM)                                     containing 5% fetal bovine serum, 2 mM L-                                     glutamate, 1× PSN (50 μg/ml penicillin, 50 μg/ml                  streptomycin, 100 μg/ml neomycin; GIBCO BRL), 10                           μM methotrexate.                                                           Serum-free Medium                                                             500 ml Dulbeccols Modified Eagle's Medium (DMEM)                              0.29 mg/ml L-glutamine                                                        10 mg/l transferrin                                                           5 mg/l fetuin (Aldrich, Milwaukee, WI)                                        5 mg/l insulin (GIBCO BRL, Grand Island, NY)                                  2 μg/l selenium (Aldrich, Milwaukee, WI)                                   In addition to the above ingredients, the medium                              was supplemented with 10 μM methotrexate, 25-50                            mM HEPES BUFFER SOLUTION (N-2-                                                Hydroxyethylpiperazine-N'-2-Ethane Sulfonic Acid                              (pH 7.2); JRH Biosciences, Lenxa, KS) and 1× PSN                        (GIBCO BRL).                                                                  Phosphate Buffered Saline (PBS)                                               8 g sodium chloride                                                           0.2 g potassium chloride                                                      1 g sodium phosphate                                                          2 g potassium phosphate                                                       Dissolve solids in distilled water. Bring                                     volume to 1 liter. Autoclave to sterilize.                                    ______________________________________                                    

To obtain a cDNA sequence encoding a protease inhibitor from the Kunitzfamily from human placenta, a human placenta cDNA library in λgt11 wasscreened using the radio-labeled ZC4792 (SEQ ID NO:5). Poly(A)⁺ RNAobtained from human placenta was used to prepare a λgt11 cDNA libraryessentially as described by Hagen et al. (Proc. Natl. Acad. Sci. USA 83:2412-241.6, 1986 and U.S. Pat. No. 4,784,950, which are incorporatedherein by reference in their entirety). The library was titered, and50,000 pfu/plate were plated on a total of twenty plates to obtain onemillion independent plaques. Duplicate plaque lifts were prepared usingICN BIOTRANS nylon membranes (ICN, Irvine, Calif.). The membranes wereprewashed in 5× SSC (Table 1), 0.5% SDS at 50° C. for one hour followedby an overnight prehybridization at 55° C. in prehybridization solution#1 (Table 1). The prehybridization solution was removed and replacedwith fresh prehybridization solution #1 containing 1.6×10⁸ cpm of ZC4792probe (SEQ ID NO:5). Hybridization was carried out under the sameconditions as the prehybridization. After hybridization, the solutionwas removed, and the blots were washed at 60° C. in 2× SSC (Table 1),0.1% SDS. Eleven positive plaques were identified and plaque purified.

Phage DNA prepared from each purified phage clone was digested with EcoRI to isolate the cDNA insert. The Eco RI fragments, which ranged insize from 2.0-3.5 kb, were then subcloned into Eco RI-linearized pUC19.Sequence analysis of the cloned fragments demonstrated that the clonesshowed some homology to the Kunitz family of protease inhibitors.Selected clones were subjected to extensive sequence analysis and wereshown to have strong homology to human amyloid protein precursorhomologue (Ponte et al., ibid.), which contains a Kunitz-type inhibitordomain. Two clones, ZGKI13 and ZGKI20, which encoded approximately 3.5kb and approximately 2.5 kb cDNA inserts, respectively, were selectedfor further analysis. Plasmids ZGKI13 and ZGKI20 were deposited on Oct.14, 1992 with the American Type Culture Collection (12301 Parklawn Dr.,Rockville, Md.) under accession numbers 69090 and 69089, respectively.Plasmids ZGKI13 and ZGKI20 were shown to contain the identical partialsequence shown in SEQ ID NOS: 1 and 2.

A comparison of the sequences of clones ZGKI13, ZGKI20 and one otherpositive clones showed that clone ZGKI13 contained a mutation at aminoacid codon 160 (from the initiation methionine) which resulted in a Trpcodon instead of a Cys codon. Clone ZGKI20 contained the correct Cyscodon at 160. A corrected cDNA constructed using the cDNAs in clonesZGKI13 and ZGKI20 was corrected and subcloned into plasmid Zem229R.Plasmid Zem229R is a mammalian expression vector modified from Zem229,which is a pUC18-based expression vector containing a unique Bam HI sitefor insertion of cloned DNA between the mouse metallothionein-1 promoterand SV40 transcription terminator and an expression unit containing theSV40 early promoter, mouse dihydroflate reductase gene, and SV40terminator. Plasmid Zem229R was deposited as an E. coli transformantwith the American Type Culture Collection (12301 Park Lawn Drive,Rockville Md.) on Sep. 28, 1993 under accession number 69447. PlasmidZem229 was modified to delete the two Eco RI sites by partial digestionwith Eco RI, blunting with DNA polymerase I (Klenow fragment) and dNTPs,and religation. Digestion of the resulting plasmid with Bam HI followedby ligation of the linearized plasmid with Bam HI-Eco RI adaptersresulted in a unique Eco RI cloning site. The resulting plasmid wasdesignated Zem229R. A plasmid containing the corrected sequence wasdesignated APPH. Clone ZGKI20 was digested with Nsi I and Bgl II toobtain the 547 bp fragment containing the correct sequence. Clone ZGKI13was digested with Eco RI and Nsi I and with Bgl II and Eco RI to isolatethe 266 bp Eco RI-Nsi I and the 2934 bp Bgl II-Eco RI fragments,respectively. The three fragments were ligated in a four-part ligationwith Eco RI-linearized Zem229R. A plasmid containing the corrected cDNAin the correct orientation relative to the metallothionein-1 promoter,designated APPH, was deposited with the American Type Culture Collection(12301 ParkLawn Drive, Rockville, Md.) on Sep. 17, 1993 under accessionnumber 69424 as an E. coli transformant. The APPH coding sequence anddeduced amino acid sequence are shown in SEQ ID NO:12 and SEQ ID NO:13.

EXAMPLE 2 Expression of Kunitz-Type Inhibitor Domains

A. Expression of a Kunitz-type Inhibitor Domain of the Amyloid PrecursorProtein Homologue Comprising Amino Acids 56 through 110 of SEQ ID NO:2

The Kunitz-type inhibitor domain encoded in plasmid. ZGKI20 andcomprising the amino acid sequence of SEQ ID NO:2 from alanine, aminoacid 56 through alanine, amino acid number 110 was expressed in a strainof the yeast Saccharomyces cerevisiae from a PCR-generated sequence. TheDNA sequence encoding the Kunitz-type inhibitor domain was amplifiedfrom human genomic DNA obtained from Clontech Laboratories, Inc. (PaloAlto, Calif.). Synthetic oligonucleotide primers M-1252 and M-1251 (SEQID NOS:7 and 6, respectively) were designed as PCR amplificationprimers. Synthetic oligonucleotide M-1252 is complementary tonucleotides 313-332 of SEQ ID NO:1, and in addition carries a 5'extension containing a translation stop codon followed by an Xba I site.Oligonucleotide M-1251 contains a sequence that is identical tonucleotides 215-235 of the synthetic leader sequence shown in SEQ IDNO:3 followed by nucleotides 168-187 of SEQ ID NO:1. A PCR reaction wasperformed in a 100 μl final volume using 1 μg of human genomic DNA(Clontech Laboratories, Inc.), 100 pmole each of oligonucleotides M-1251and M-1252 (SEQ ID NOS:6 and 7, respectively), and the reagents providedin the GENEAMP kit (Perkin Elmer Cetus, Norwalk, Conn.) according to themanufacturer's instructions. The reaction was amplified for nineteencycles (20 seconds at 94° C., 20 seconds at 50° C., and 30 seconds at72° C.) followed by a ten minute incubation at 72° C. A 201 bp fragmentwas isolated by agarose gel electrophoresis.

A DNA sequence encoding the synthetic signal sequence (SEQ ID NO:3) wasobtained by PCR amplification of a fragment from plasmid pKFN-1000.Plasmid pKFN-1000 is a derivative of plasmid pTZ19R (Mead et al., Prot.Engin. 1: 67-74, 1986) containing a DNA sequence encoding a syntheticyeast signal leader peptide. Plasmid pKFN-1000 is described in WO90/10075, which is incorporated by reference herein in its entirety. TheDNA sequence of the 235 base pairs downstream from the Eco RI site ofplasmid pKFN-1000 and the encoded amino acid sequence is shown in SEQ IDNOS:3 and 4. A 0.7 kb Pvu II fragment of plasmid pKFN-1000 was used as atemplate. Synthetic oligonucleotide NOR-1478 (SEQ ID NO:8) is identicalto a sequence just upstream of the Eco RI site (nucleotides to 1-6 ofSEQ ID NO:3). Synthetic oligonucleotide NOR-2523 (SEQ ID NO:9) iscomplementary to nucleotides 215-235 of the coding sequence in SEQ IDNO:3. A PCR reaction was performed in a 100 μl final volume using 0.1 μgof the 0.7 kb Pvu II fragment, 100 pmoles each of oligonucleotideNOR-1478 and NOR-2523 (SEQ ID NOS:8 and 9, respectively) and reagentsfrom the GENEAMP commercial kit (Perkin Elmer Cetus) according to themanufacturer's instructions. The PCR reaction was amplified as describedabove. A 257 bp PCR product was isolated by agarose gel electrophoresis.

A DNA sequence encoding the complete synthetic signal sequenceoperatively linked to the Kunitz-type inhibitor domain sequence wasobtained by amplifying the two PCR fragments described above. A PCRreaction was performed as described above using 100 pmoles each ofprimers NOR-1478 (SEQ ID NO:8) and M-1252 (SEQ ID NO:7) and 0.1 μg ofeach of the two PCR fragments described above. The PCR reaction wasamplified for sixteen cycles (1 minute at 94° C., 2 minutes at 50° C., 3minutes at 71° C.) followed by a ten minute incubation at 72° C. A 437bp fragment was purified by agarose gel electrophoresis. The fragmentwas then digested with Eco RI and Xba I, and the resulting 404 bpfragment was ligated with plasmid pTZ19R, which had been linearized bydigestion with Eco RI and Xba I. The ligation mixture was transformedinto competent restriction minus, modification plus E. coli strain, andtransformants were selected in the presence of ampicillin. Plasmid DNAsprepared from selected transformants were sequenced, and a plasmidcontaining the DNA sequence of the synthetic yeast signal sequence fusedto the Kunitz-type inhibitor domain was identified.

The Eco RI-Xba I fragment encoding the secretory signal-Kunitz-typeinhibitor domain was then isolated and subcloned into plasmid pMT-636.Plasmid pMT-636 is derived from the shuttle vector pCPOT (Plasmid pCPOTwas deposited on May 9, 1984 with the American Type Culture Collection;12301 Parklawn Dr., Rockville, Md.; under Accession No. 39685) in whichthe 0.4 kb Hpa I-Nru I fragment containing the Saccharomyces cerevisiaeLEU2 gene was deleted and, in addition, contains the Saccharomycescerevisiae TPI1 promoter and the TPI1 terminator flanking an Eco RI-XbaI directional cloning site such that the a DNA insert is transcribed inthe same direction as the Schizosaccharomyces pombe POT1 gene (Norris etal., ibid.). Plasmid pMT-636 has been described in WO 89/01968 and WO90/10075, which are incorporated herein by reference in their entirety.Plasmid pMT-636 was digested with Nco I and Xba I to isolate the 9.3 kbfragment. Plasmid pMT-636 was also digested with Nco I and Eco RI toobtaion the 1.6 kb fragment. The two fragments from pMT-636 were ligatedwith the Eco RI-Xba I fragment.

A plasmid containing the signal sequence-Kunitz-type inhibitor domainfragment in the correct orientation was transformed into S. cerevisiaeMT-663 (a/α Δtpi/Δtpi pep4-3/pep4-3). Transformants were selected forgrowth on glucose as the sole carbon source, and cultivated in YEPDmedia. Transformants were assayed for activity as described in Example3. The Kunitz-type inhibitor is purified as described in Example 4.

B. Expression of a Kunitz-type Inhibitor Domain of the Amyloid PrecursorProtein Homologue Comprising Amino Acids 53 through 110 of SEQ ID NO:2

A DNA construct encoding a Kunitz-type inhibitor domain the Kunitz-typeinhibitor domain comprising the amino acid sequence of SEQ ID NO:2 fromaspartic acid, amino acid number 53, through alanine, amino acid number110, was amplified from human genomic DNA as described in Example 1using oligonucleotide primers M-1252 and M-1249 (SEQ ID NOS:7 and 10).The resulting PCR-generated fragment was gel-purified and joined to thesignal sequence as described above. The fragment encoding the signalsequence-Kunitz-type inhibitor was then subcloned into a yeastexpression vector and transformed into Saccharomyces cerevisiae strainMT-663 as described above.

Selected transformants were assayed for activity as described in Example3. The Kunitz-type inhibitor is purified as described in Example 4.

C. Expression of a Kunitz-type Inhibitor Domain of the Amyloid PrecursorProtein Homologue Comprising A Glutamic Acid Residue Followed by AminoAcids 57 through 110 of SEQ ID NO:2

A DNA construct encoding a Kunitz-type inhibitor comprising the aminoacid sequence of SEQ ID NO:2 from valine, amino acid number 57, throughalanine, amino acid number 110, and which further contains a glutamicacid immediately N-terminal to valine was amplified from human genomicDNA as described in Example 1 using oligonucleotide primers M-1252 andM-1250 (SEQ ID NOS:7 and 11). The resulting PCR-generated fragment wasgel-purified and joined to the signal sequence as described above. Thefragment encoding the signal sequence-Kunitz-type inhibitor was thensubcloned into a yeast expression vector and transformed intoSaccharomyces cerevisiae strain MT-663 as described above.

Selected transformants were assayed for activity as described in Example3. The Kunitz-type inhibitor is purified as described in Example 4.

D. Expression of the Amyloid Precursor Protein Homologue in MammalianCells

Plasmid APPH DNA, encoding the amyloid precursor protein homologue wasused to transfect BHK570 cells using calcium phosphate-mediatedtransfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson,Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973). BHK 570 cells were deposited with the American Type CultureCollection (ATCC; 12301 Parklawn Dr., Rockville, Md., 20852, USA) onDec. 20, 1989 under accession number CRL 10314. Transfected cells wereinitially selected in the presence of medium containing 1 μM ofmethotrexate followed by more stringent selection in medium containing10 μM methotrexate. Following selection in 10 μM methotrexate, randomlyselected clones were grown to confluency in 6-well dishes in GrowthMedium (Table 1). After reaching confluency, the spent medium wasdecanted, and the cells were washed with PBS (Phosphate Buffered Saline;Table 1) to remove any remaining serum. Serum-free medium (Table 1) wasadded to the cells, and the cells were grown for 24-48 hours. Theconditioned media was collected and assayed for trypsin inhibitoractivity using the assay method detailed in Example 4B.

E. Expression of a Kunitz-type Inhibitor domain of the Amyloid PrecursorProtein

The Kunitz-type inhibitor domain of the amyloid protein precursor (SEQID NO:17) was expressed in a strain of the yeast Saccharomycescerevisiae from a PCR-generated sequence. The DNA sequence encoding theKunitz-type inhibitor domain was amplified from human genomic DNAobtained from Clontech Laboratories, Inc. (Palo Alto, Calif.)essentially as described above. Synthetic oligonucleotide primers primer1 and primer 2 (SEQ ID NOS:14 and 15, respectively) were designed as PCRamplification primers. Synthetic oligonucleotide primer 2 (SEQ ID NO:15)is complementary to nucleotides 151-168 of SEQ ID NO:16, and in additioncarries a 5' extension containing a translation stop codon followed byan Xba I site. Oligonucleotide primer 1 (SEQ ID NO:14) contains asequence that is identical to nucleotides 215-235 of the syntheticleader sequence shown in SEQ ID NO:3 followed by nucleotides 1-16 of SEQID NO:16. A PCR reaction was performed in a 100 μl final volume using 1μg of human genomic DNA (Clontech Laboratories, Inc.), 100 pmole each ofoligonucleotides primer 1 and primer 2 (SEQ ID NOS:14 and 15,respectively), and the reagents provided in the GENEAMP kit (PerkinElmer Cetus, Norwalk, Conn.) according to the manufacturer'sinstructions. The reaction was amplified for nineteen cycles (20 secondsat 94° C., 20 seconds at 50° C. and 30 seconds at 72° C.) followed by aten minute incubation at 72° C. A 201 bp fragment was isolated byagarose gel electrophoresis.

A DNA sequence encoding the synthetic signal sequence (SEQ ID NO:3) wasobtained by PCR amplification of a fragment from plasmid pKFN-1000 asdescribed above. A DNA sequence encoding the complete synthetic signalsequence operatively linked to the Kunitz-type inhibitor domain sequencewas obtained by amplifying the two PCR fragments described above. A PCRreaction was performed as described above using 100 pmoles each ofprimers NOR-1478 (SEQ ID NO:8) and primer 2 (SEQ ID NO:15) and 0.1 μg ofeach of the two PCR fragments described above. The PCR reaction wasamplified for sixteen cycles (1 minute at 94° C., 2 minutes at 50° C., 3minutes at 71° C.) followed by a ten minute incubation at 72° C. A 437bp fragment was purified by agarose gel electrophoresis. The fragmentwas then digested with Eco RI and Xba I, and the resulting fragment wasligated with plasmid pTZ19R as described above. The ligation mixture wastransformed into competent restriction minus, modification plus E. colistrain, and transformants were selected in the presence of ampicillin.Plasmid DNAs prepared from selected transformants were sequenced, and aplasmid containing the DNA sequence of the synthetic yeast signalsequence fused to the APP Kunitz-type inhibitor domain was identified.

The Eco RI-Xba I fragment encoding the secretory signal-Kunitz-typeinhibitor domain was then isolated and subcloned into plasmid pMT-636 asdescribed above. A plasmid containing the signal sequence-APPKunitz-type inhibitor domain fragment in the correct orientation wastransformed into S. cerevisiae MT-663 (a/α Δtpi/Δtpi pep4-3/pep4-3).Transformants were selected for growth on glucose as the sole carbonsource, and cultivated in YEPD media. Transformants were assayed foractivity as described in Example 3. The amyloid precursor proteinKunitz-type inhibitor was purified as described in Example 4.

Example 3 Activity Assays

A. Trypsin Inhibitory Activity Assay on Yeast Culture Supernatants

Trypsin inhibitory activity was measured on the spent media fromcultures of yeast transformants described in Example 2 4 by diluting 3.2μl of each spent medium sample with 80 μl of assay buffer (50 mM TrisHCl, pH 7.4, 100 mM NaCl, 2 mM CaCl₂, 0.1% w/v PEG 20,000). The dilutedsupernatant was added to 80 μl of 133 nM bovine trypsin (Novo NordiskA/S) diluted in assay buffer, and the mixture was incubated for 10minutes at room temperature. After incubation, 100 μl of 1.8 mM peptidylnitroanilide substrate S2251 (D-Val-Leu-Lys-Nan; Kabi) diluted in assaybuffer was added to each sample, and the samples were incubated with thesubstrate for 30 minutes. Trypsin inhibitory activity, as indicated by acolorless solution, was found in supernatants from the yeast strainsdescribed in Example 2. A control reaction, which resulted in a yellowsolution, was produced by a supernatant from a yeast strain notexpressing any Kunitz-type inhibitor.

B. Trypsin Inhibitory Activity Assay on Mammalian Cell CultureSupernatants

Conditioned media from cells expressing Kunitz-type inhibitors wasassayed for trypsin inhibitor activity. For each clone, 20-100 μl ofconditioned medium was added to a solution containing 2.4 μg/ml trypsin(Worthington Biochemical, Freehold, N.J.) in 100 mM NaCl, 50 mM Tris (pH7.4) to give a final volume of 300 μl . The reactions were incubated at23° C. for 30 minutes after which the chromogenic substrate S-2251(D-Val-Leu-Lys-Nan; Chromogenix, Franklin, Ohio) to a finalconcentration of 0.6 mM. The residual trypsin activity was measured byabsorbance at 405 nm.

C. Protease Inhibitory Activity Assays

Protease inhibitory profiles of the Kunitz inhibitors were determinedfor a variety of proteases using the chromogenic substrates listed inTable 2 and compared to the inhibitory activity shown by the Kunitz-typeinhibitor domain of the amyloid protein precursor (Example 2E) andbovine aprotinin (amino acids 1-58 as described by Norris et al., ibid.;which is incorporated by reference herein in its entirety).

                                      TABLE 2                                     __________________________________________________________________________    Protease (concentration)                                                                       Substrate (concentration)                                    Source           Source                                                       __________________________________________________________________________    Trypsin (8 nM)   H--D-Val--Leu--Lys--pNA (0.6 mM)                             Novo Nordisk A/S,                                                                              Kabi                                                         Bagsvaerd, Denmark                                                            Chymotrypsin (2.5 nM)                                                                          MeO--Suc--Arg--Pro--Tyr--pNA (0.6 mM)                        Novo Nordisk A/S Kabi                                                         GL Kallikrein (1 U/ml)                                                                         H--D-Val--Leu--Arg--pNA (0.6 mM)                             Sigma, St Louis, MO                                                                            Kabi                                                         Plasmin (10 nM)  H--D-Val--Leu--Lys--pNA (0.6 mM)                             Kabi             Kabi                                                         Urokinase (5 nM) <Glu--Gly--Arg--pNA (0.6 mM)                                 Serono           Kabi                                                         Freigurg, Germany                                                             rec. Protein Ca (5 nM)                                                                         <Glu--Pro--Arg--pNA (0.6 mM)                                 Novo Nordisk A/S Kabi                                                         PL Kallikrein (3 nM)                                                                           H--D-Pro--Phe--Arg--pNA (0.6 mM)                             Kabi             Kabi                                                         human Factor XIIa (30 nM)                                                                      H--D-Pro--Phe--Arg--pNA (0.6 mM)                             Dr. Walt Kisiel  Kabi                                                         University of New Mexico,                                                     Albuquerque, NM                                                               human Factor XIa (1 nM)                                                                        Boc--Glu(OBzl)--Ala--Arg--MCA (0.12 mM)                      Dr. Kazoo Fujikawa                                                                             Peptide Institute                                            University of Washington,                                                                      Osaka, Japan                                                 Seattle, WA                                                                   human Factor Xa (3 nM)                                                                         MeO--CO--CHA--Gly--Arg--pNA (0.3 mM)                         Dr. I. Schousboe NycoMed                                                      Copenhagen, Denmark                                                                            Oslo, Norway                                                 rec. human Factor VIIa (300 nM)                                                                H--D-lie--Pro--Arg--pNA (0.6 mM)                             Novo Nordisk A/S Kabi                                                         Leukocyte Elastase                                                                             MeO--Suc--Ala--Ala--Pro--Val--pNA (0.6 mM)                   purified at Novo Nordisk A/S                                                                   (SEQ ID NO: 18)                                              using the method of                                                                            Sigma Chemical Co.                                           Baugh and Travis St. Louis, MO                                                (Biochemistry 15: 836-843, 1976)                                              Cathepsin G      Suc--Ala--Ala--Pro--Phe--pNA (0.6 mM)                        purified at Novo Nordisk A/S                                                                   (SEQ ID NO: 19)                                              using the method of                                                                            Sigma Chemical Co.                                           Baugh and Travis                                                              (Biochemistry 15: 836-843, 1976)                                              __________________________________________________________________________     Abbreviations in Table 2: rec. refers to recombinant, GL kallikrein refer     to glandular kallikrein, and PL kallikrein refers to plasma kallikrein.  

Inhibition assays were performed in microtiter wells in a total volumeof 300 μl in 10 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.01% TWEEN80(Polyoxyethylenesorbitan monoleate). Each reaction contained 1 μM of thesample inhibitor and one of the proteases at the concentration listed inTable 2. The reactions were incubated at 25° C. for ten minutes afterwhich the appropriate chromogenic substrate was added to the finalconcentration listed in Table 2 and the final reaction was incubated forthirty minutes at 25° C. Amidolytic activity was measured at 405 nm orby fluorescence Em at 460 nm. Percent inhibition was determined relativeto reactions carried out in the absence of inhibitor representing 100%activity or 0% inhibition. The results of the assay are shown in Table3.

                  TABLE 3                                                         ______________________________________                                                    % Inhibition                                                      Protease      APPH        APP     BPTI                                        ______________________________________                                        Trypsin       99.6        99.7    100                                         Chymotrypsin  61          94      91.3                                        Gl Kallikrein 97          82      91.4                                        Plasmin       77          90      100                                         Urokinase     2           0       1                                           r.h. Protein Ca                                                                             0           1       38                                          PL Kallikrein 69          71      83                                          h. Factor XIIa                                                                              6           8       2                                           h. Factor XIa 99.8        100     59                                          h. Factor Xa  0           29      1                                           r.h. Factor VIIa                                                                            15          7       1                                           Leukocyte Elastase                                                                          4           6       1                                           Cathepsin G   0           16      4                                           ______________________________________                                         The abbreviations in Table 3: r.h. refers to recombinant human, h. refers     to human, protein Ca refers to activated protein C; APPH refers to the        KunitzType inhibitor domain of Example 2E, APP refers to the amylold          protein precursor KunitzType inhibitor domain (Example 2E) and BPTI refer     to bovine aprotinin (amino acids 1-58).                                  

EXAMPLE 4 Purification of Kunitz-Type Inhibitors

Kunitz-type inhibitors are purified essentially as described by Norriset al. (ibid.; which is incorporated herein by reference). Selectedtransformants are grown in 10 liters of YEPD for approximately four daysat 30° C. until an OD₆₀₀ of approximately 30 had been reached. At theend of the fermentation, the pH of the culture was adjusted to 3.0 bythe addition of concentrated H₃ PO₄. The culture was centrifuged, andthe supernatant was decanted.

For purification, approximately 1 liter of supernatant was adjusted topH 8.0 by the addition of solid Tris-HCl to a final concentration of 50mM and titration with 4M NaOH. The supernatant was filtered before itwas applied to a column of bovine trypsin that adsorbed toCNBr-activated Sepharose (350 mg bovine trypsin per 35 ml gel). Thecolumn was washed with 150 ml 0.1 Tris-HCl (pH 8.0), 0.5M NaCl followedby 150 ml 0.01M Tris-HCl (pH 8.0). After the 0.01M Tris-HCl (pH 8.0)wash, the bound material was eluted with 200 ml 0.2M glycine-HCl (pH3.0). Fractions of 10 ml each were collected and were analyzed byreverse phase HPLC. Protein-containing fractions were then pooled.

The pooled material was applied to a preparative reverse phase HPLCcolumn (Vydac, The Separations Group, Hesperia, Calif. or the like) thathad been equilibrated with 5% B (0.7% TFA in acetonitrile) and 95% A(0.1% TFA in H₂ O). The flow rate across the column was maintained at 4ml/min. Following sample application, the column was washed with 5% Buntil a baseline at 214 nm was achieved. Gradient elution with fractioncollection was performed from 5 to 85% B over 80 min. Fractionscontaining UV-absorbing material were analyzed by reverse phase HPLC(Vydac) and were combined to give pools of chromatographically purematerial. Solvent was removed from the pooled fractions by vacuumcentrifugation. The concentration and total yield of inhibitor in themajor pools was estimated by reverse phase HPLC analysis and bycomparison to a bovine aprotinin standard. The final preparations werecharacterized by electronspray mass spectroscopy (SCIEX API III or thelikeu).

Kunitz inhibitor activity is measured using the method essentiallydescribed by Norris et al. (ibid.). Briefly, various fixedconcentrations of the Kunitz-type inhibitor are incubated in thepresence of 0.24 μg/ml of porcine trypsin (Novo Nordisk A/S, Bagsvaerd,Denmark), 12.8 CU/l human plasmin (Kabi, Stockholm, Sweden) or 0.16nkat/ml human plasma kallikrein (Kabi) in 100 mM NaCl, 50 mM Tris HCl7.4. After a 30 minute incubation the residual enzymatic activity ismeasured by the degradation of a solution containing 0.6 mM of either ofthe chromogenic peptidyl nitroanilide trypsin/plasmin substrates S2251(D-Val-Leu-Lys-Nan; Kabi) or S2302 (D-Pro-Phe-Arg-Nan; Kabi) in assaybuffer. The samples are incubated for 30 minutes after which theabsorbance of each sample is measured at 405 nm. Plasmin or trypsinactivity is measured as a decrease in absorbance at 405 nm. From theresults, the apparent inhibition constant Ki is calculated.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 19                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 399 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vii) IMMEDIATE SOURCE:                                                       (B ) CLONE: ZGKI13 and ZGKI20                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..398                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CAGCTGTGGATGAGGATGATGAGGATGAGGAAGAAGGGGAGGAAGTG47                             AlaValAspGluAspAspGluAspGluGluGluGlyGluG luVal                                151015                                                                        GTGGAGGACCGAGATTACTACTATGACACCTTCAAAGGAGATGACTAC95                            ValGluAspArgAspTyrTyrTyrAspThrPheLysGlyA spAspTyr                             202530                                                                        AATGAGGAGAATCCTACTGAACCCGGCAGCGACGGCACCATGTCAGAC143                           AsnGluGluAsnProThrGluProGlySerAspGlyThr MetSerAsp                             354045                                                                        AAGGAAATTACTCATGATGTCAAAGCTGTCTGCTCCCAGGAGGCGATG191                           LysGluIleThrHisAspValLysAlaValCysSerGlnGlu AlaMet                             505560                                                                        ACGGGGCCCTGCCGGGCCGTGATGCCTCGTTGGTACTTCGACCTCTCC239                           ThrGlyProCysArgAlaValMetProArgTrpTyrPheAspLeuSe r                             657075                                                                        AAGGGAAAGTGCGTGCGCTTTATATATGGTGGCTGCGGCGGCAACAGG287                           LysGlyLysCysValArgPheIleTyrGlyGlyCysGlyGlyAsnArg                              80 859095                                                                     AACAATTTTGAGTCTGAGGATTATTGTATGGCTGTGTGTAAAGCGATG335                           AsnAsnPheGluSerGluAspTyrCysMetAlaValCysLysAlaMet                               100105110                                                                    ATTCCTCCAACTCCTCTGCCAACCAATGATGTTGATGTGTATTTCGAG383                           IleProProThrProLeuProThrAsnAspValAspValTyrPheGlu                               115120125                                                                    ACCTCTGCAGATGATA399                                                           ThrSerAlaAspAsp                                                               130                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 132 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaValAspGluAspAspGluAspGluGluGluGlyGluGluValVal                              151015                                                                         GluAspArgAspTyrTyrTyrAspThrPheLysGlyAspAspTyrAsn                             202530                                                                        GluGluAsnProThrGluProGlySerAspGlyThrMetSerAspLys                              35 4045                                                                       GluIleThrHisAspValLysAlaValCysSerGlnGluAlaMetThr                              505560                                                                        GlyProCysArgAlaValMetProArgTrpTy rPheAspLeuSerLys                             65707580                                                                      GlyLysCysValArgPheIleTyrGlyGlyCysGlyGlyAsnArgAsn                              8590 95                                                                       AsnPheGluSerGluAspTyrCysMetAlaValCysLysAlaMetIle                              100105110                                                                     ProProThrProLeuProThrAsnAspValAspValTyrPheGluT hr                             115120125                                                                     SerAlaAspAsp                                                                  130                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 235 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                            (B) LOCATION: 77..235                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                ATAAACGACCAAAAGAATGAAGGCTGTTTTCTTGGTTTTGTCCTTGATC109                           MetLysAlaValPheLeuValLeuSerLeuIle                                            1510                                                                          GGATTCTGCTGGGCCCAACCAGTCACTGGCGATGAATCATCTGTTGAG157                           GlyPheCy sTrpAlaGlnProValThrGlyAspGluSerSerValGlu                             152025                                                                        ATTCCGGAAGAGTCTCTGATCATCGCTGAAAACACCACTTTGGCTAAC205                           IleProGluG luSerLeuIleIleAlaGluAsnThrThrLeuAlaAsn                             303540                                                                        GTCGCCATGGCTGAGAGATTGGAGAAGAGA235                                             ValAlaMetAlaGlu ArgLeuGluLysArg                                               4550                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetLysAlaValPheLeuValLeuSerLeu IleGlyPheCysTrpAla                             151015                                                                        GlnProValThrGlyAspGluSerSerValGluIleProGluGluSer                              2025 30                                                                       LeuIleIleAlaGluAsnThrThrLeuAlaAsnValAlaMetAlaGlu                              354045                                                                        ArgLeuGluLysArg                                                               50                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC4792                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GTTGTTGCTGTTGCCTCCGCAGCCTCCGTA30                                              (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 42 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: M-1251                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCTGAGAGATTGGGAGAAGAGAGCTGTCTGCTCCCAGGAGGC42                                  (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: M-1252                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AGTTGGTCTAGATTACGCTTTACACACAGCCATAC 35                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: NOR-1478                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GTAAAACGACGGCCAGT 17                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: NOR-2523                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCTCTTCTCCAATCTCTCAGC 21                                                      (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: M-1249                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCTGAGAGATTG GAGAAGAGAGATGTCAAAGCTGTCTGCTCCC43                                (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: M-1250                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCTGAGAGATTGGAGAAGAGAGAAGTCTGCTCCCAGGAGGC41                                   (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3725 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapiens                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: APPH                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 73..2364                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GTCGCGGTGTGCTAAGCGAGGAGTCCGAGTGTGTGAGCTTGAGAGCCGCGCGCTAGAGCG60                ACCCGGCGAGGG ATGGCGGCCACCGGGACCGCGGCCGCCGCAGCCACG108                          MetAlaAlaThrGlyThrAlaAlaAlaAlaAlaThr                                          1510                                                                          GGCAGGCTCCTGCTT CTGCTGCTGGTGGGGCTCACGGCGCCTGCCTTG156                          GlyArgLeuLeuLeuLeuLeuLeuValGlyLeuThrAlaProAlaLeu                              152025                                                                        GCGCTGGCCGGCTACATCGA GGCTCTTGCAGCCAATGCCGGAACAGGA204                          AlaLeuAlaGlyTyrIleGluAlaLeuAlaAlaAsnAlaGlyThrGly                              303540                                                                        TTTGCTGTTGCTGAGCCTCAAATCGCAA TGTTTTGTGGGAAGTTAAAT252                          PheAlaValAlaGluProGlnIleAlaMetPheCysGlyLysLeuAsn                              45505560                                                                      ATGCATGTGAACATTCAGACTGGG AAATGGGAACCTGATCCAACAGGC300                          MetHisValAsnIleGlnThrGlyLysTrpGluProAspProThrGly                              657075                                                                        ACCAAGAGCTGCTTTGAAACAAAA GAAGAAGTTCTTCAGTACTGTCAG348                          ThrLysSerCysPheGluThrLysGluGluValLeuGlnTyrCysGln                              808590                                                                        GAGATGTATCCAGAGCTACAGATCAC AAATGTGATGGAGGCAAACCAG396                          GluMetTyrProGluLeuGlnIleThrAsnValMetGluAlaAsnGln                              95100105                                                                      CGGGTTAGTATTGACAACTGGTGCCGGAGGG ACAAAAAGCAATGCAAG444                          ArgValSerIleAspAsnTrpCysArgArgAspLysLysGlnCysLys                              110115120                                                                     AGTCGCTTTGTTACACCTTTCAAGTGTCTCGTGGGTGAA TTTGTAAGT492                          SerArgPheValThrProPheLysCysLeuValGlyGluPheValSer                              125130135140                                                                  GATGTCCTGCTAGTTCCAGAAAAGTGCCAGTTTTTC CACAAAGAGCGG540                          AspValLeuLeuValProGluLysCysGlnPhePheHisLysGluArg                              145150155                                                                     ATGGAGGTGTGTGAGAATCACCAGCACTGGCACAC GGTAGTCAAAGAG588                          MetGluValCysGluAsnHisGlnHisTrpHisThrValValLysGlu                              160165170                                                                     GCATGTCTGACTCAGGGAATGACCTTATATAGCTACG GCATGCTGCTC636                          AlaCysLeuThrGlnGlyMetThrLeuTyrSerTyrGlyMetLeuLeu                              175180185                                                                     CCATGTGGGGTAGACCAGTTCCATGGCACTGAATATGTGTGC TGCCCT684                          ProCysGlyValAspGlnPheHisGlyThrGluTyrValCysCysPro                              190195200                                                                     CAGACAAAGATTATTGGATCTGTGTCAAAAGAAGAGGAAGAGGAAGAT 732                          GlnThrLysIleIleGlySerValSerLysGluGluGluGluGluAsp                              205210215220                                                                  GAAGAGGAAGAGGAAGAGGAAGATGAAGAGGAAGACTATGATGTTTA T780                          GluGluGluGluGluGluGluAspGluGluGluAspTyrAspValTyr                              225230235                                                                     AAAAGTGAATTTCCTACTGAAGCAGATCTGGAAGACTTCACAGAAG CA828                          LysSerGluPheProThrGluAlaAspLeuGluAspPheThrGluAla                              240245250                                                                     GCTGTGGATGAGGATGATGAGGATGAGGAAGAAGGGGAGGAAGTGGTG 876                          AlaValAspGluAspAspGluAspGluGluGluGlyGluGluValVal                              255260265                                                                     GAGGACCGAGATTACTACTATGACACCTTCAAAGGAGATGACTACAAT92 4                          GluAspArgAspTyrTyrTyrAspThrPheLysGlyAspAspTyrAsn                              270275280                                                                     GAGGAGAATCCTACTGAACCCGGCAGCGACGGCACCATGTCAGACAAG972                           GluGlu AsnProThrGluProGlySerAspGlyThrMetSerAspLys                             285290295300                                                                  GAAATTACTCATGATGTCAAAGCTGTCTGCTCCCAGGAGGCGATGACG1020                          Glu IleThrHisAspValLysAlaValCysSerGlnGluAlaMetThr                             305310315                                                                     GGGCCCTGCCGGGCCGTGATGCCTCGTTGGTACTTCGACCTCTCCAAG1068                          Gl yProCysArgAlaValMetProArgTrpTyrPheAspLeuSerLys                             320325330                                                                     GGAAAGTGCGTGCGCTTTATATATGGTGGCTGCGGCGGCAACAGGAAC1116                          GlyL ysCysValArgPheIleTyrGlyGlyCysGlyGlyAsnArgAsn                             335340345                                                                     AATTTTGAGTCTGAGGATTATTGTATGGCTGTGTGTAAAGCGATGATT1164                          AsnPheGlu SerGluAspTyrCysMetAlaValCysLysAlaMetIle                             350355360                                                                     CCTCCAACTCCTCTGCCAACCAATGATGTTGATGTGTATTTCGAGACC1212                          ProProThrProLeuPro ThrAsnAspValAspValTyrPheGluThr                             365370375380                                                                  TCTGCAGATGATAATGAGCATGCTCGCTTCCAGAAGGCTAAGGAGCAG1260                          SerAlaAspAspAs nGluHisAlaArgPheGlnLysAlaLysGluGln                             385390395                                                                     CTGGAGATTCGGCACCGCAACCGAATGGACAGGGTAAAGAAGGAATGG1308                          LeuGluIleArgH isArgAsnArgMetAspArgValLysLysGluTrp                             400405410                                                                     GAAGAGGCAGAGCTTCAAGCTAAGAACCTCCCCAAAGCAGAGAGGCAG1356                          GluGluAlaGluLeu GlnAlaLysAsnLeuProLysAlaGluArgGln                             415420425                                                                     ACTCTGATTCAGCACTTCCAAGCCATGGTTAAAGCTTTAGAGAAGGAA1404                          ThrLeuIleGlnHisPheGln AlaMetValLysAlaLeuGluLysGlu                             430435440                                                                     GCAGCCAGTGAGAAGCAGCAGCTGGTGGAGACCCACCTGGCCCGAGTG1452                          AlaAlaSerGluLysGlnGlnLeuValGl uThrHisLeuAlaArgVal                             445450455460                                                                  GAAGCTATGCTGAATGACCGCCGTCGGATGGCTCTGGAGAACTACCTG1500                          GluAlaMetLeuAsnAspArgArgA rgMetAlaLeuGluAsnTyrLeu                             465470475                                                                     GCTGCCTTGCAGTCTGACCCGCCACGGCCTCATCGCATTCTCCAGGCC1548                          AlaAlaLeuGlnSerAspProPro ArgProHisArgIleLeuGlnAla                             480485490                                                                     TTACGGCGTTATGTCCGTGCTGAGAACAAAGATCGCTTACATACCATC1596                          LeuArgArgTyrValArgAlaGluAsn LysAspArgLeuHisThrIle                             495500505                                                                     CGTCATTACCAGCATGTGTTGGCTGTTGACCCAGAAAAGGCGGCCCAG1644                          ArgHisTyrGlnHisValLeuAlaValAspPr oGluLysAlaAlaGln                             510515520                                                                     ATGAAATCCCAGGTGATGACACATCTCCACGTGATTGAAGAAAGGAGG1692                          MetLysSerGlnValMetThrHisLeuHisValIleGluG luArgArg                             525530535540                                                                  AACCAAATCCTCTCTCTGCTCTACAAAGTACCTTATGTAGCCCAAGAA1740                          AsnGlnIleLeuSerLeuLeuTyrLysValProTyr ValAlaGlnGlu                             545550555                                                                     ATTCAAGAGGAAATTGATGAGCTCCTTCAGGAGCAGCGTGCAGATATG1788                          IleGlnGluGluIleAspGluLeuLeuGlnGluGln ArgAlaAspMet                             560565570                                                                     GACCAGTTCACTGCCTCAATCTCAGAGACCCCTGTGGACGTCCGGGTG1836                          AspGlnPheThrAlaSerIleSerGluThrProValAs pValArgVal                             575580585                                                                     AGCTCTGAGGAGAGTGAGGAGATCCCACCGTTCCACCCCTTCCACCCC1884                          SerSerGluGluSerGluGluIleProProPheHisProPheH isPro                             590595600                                                                     TTCCCAGCCCTACCTGAGAACGAAGACACTCAGCCGGAGTTGTACCAC1932                          PheProAlaLeuProGluAsnGluAspThrGlnProGluLeuTyrHis                              605 610615620                                                                 CCAATGAAAAAAGGATCTGGAGTGGGAGAGCAGGATGGGGGACTGATC1980                          ProMetLysLysGlySerGlyValGlyGluGlnAspGlyGlyLeuIle                              625630635                                                                     GGTGCCGAAGAGAAAGTGATTAACAGTAAGAATAAAGTGGATGAAAAC2028                          GlyAlaGluGluLysValIleAsnSerLysAsnLysValAspGluAs n                             640645650                                                                     ATGGTCATTGACGAGACTCTGGATGTTAAGGAAATGATTTTCAATGCC2076                          MetValIleAspGluThrLeuAspValLysGluMetIlePheAsnAla                               655660665                                                                    GAGAGAGTTGGAGGCCTCGAGGAAGAGCGGGAATCCGTGGGCCCACTG2124                          GluArgValGlyGlyLeuGluGluGluArgGluSerValGlyProLeu                              670 675680                                                                    CGGGAGGACTTCAGTCTGAGTAGCAGTGCTCTCATTGGCCTGCTGGTC2172                          ArgGluAspPheSerLeuSerSerSerAlaLeuIleGlyLeuLeuVal                              685 690695700                                                                 ATCGCAGTGGCCATTGCCACGGTCATCGTCATCAGCCTGGTGATGCTG2220                          IleAlaValAlaIleAlaThrValIleValIleSerLeuValMetLeu                               705710715                                                                    AGGAAGAGGCAGTATGGCACCATCAGCCACGGGATCGTGGAGGTTGAT2268                          ArgLysArgGlnTyrGlyThrIleSerHisGlyIleValGluValAsp                               720725730                                                                    CCAATGCTCACCCCAGAAGAGCGTCACCTGAACAAGATGCAGAACCAT2316                          ProMetLeuThrProGluGluArgHisLeuAsnLysMetGlnAsnHis                              735 740745                                                                    GGCTATGAGAACCCCACCTACAAATACCTGGAGCAGATGCAGATTTAGGTGGCAG                       2371                                                                          GlyTyrGluAsnProThrTyrLysTyrLeuGluGlnMetGlnIle                                 750 755760                                                                    GGAGCGCGGCAGCCCTGGCGGAGGGATGCAGGTGGGCCGGAAGATCCCACGATTCCGATC2431              GACTGCCAAGCAGCAGCCGCTGCCAGGGGCTGCGTCTGACATCCTGACCTCCTGGACTGT2491              AGGACTATATAAAGTACTAC TGTAGAACTGCAATTTCCATTCTTTTAAATGGGTGAAAAA2551             TGGTAATATAACAATATATGATATATAAACCTTAAATGAAAAAAATGATCTATTGCAGAT2611              ATTTGATGTAGTTTTCTTTTTTAAATTAATCAGAAACCCCACTTCCATTGTATTGTCTGA2671              CACATGCTCTCAATATATAATAAATGGGAAATGTCGATTTTCAATAATAGACTTATATGC2731              AGGCTGTCGTTCCGGTTATGTTGTGTAAGTCAACTCTTCAGCCTCATTCACTGTCCTGGC2791              TTTTATTTAAAGAAAAAAAAGGCAGTATTCCCTTTTTAAATGA GCTTTCAGGAAGTTGCT2851             GAGAAATGGGGTGGAATAGGGAACTGTAATGGCCACTGAAGCACGTGAGAGACCCTCGCA2911              AAATGATGTGAAAGGACCAGTTTCTTGAAGTCCAGTGTTTCCACGGCTGGATACCTGTGT2971              GTCTCCATAAAAGTCCTGTC ACCAAGGACGTTAAAGGCATTTTATTCCAGCGTCTTCTAG3031             AGAGCTTAGTGTATACAGATGAGGGTGTCCGCTGCTGCTTTCCTTCGGAATCCAGTGCTT3091              CCACAGAGATTAGCCTGTAGCTTATATTTGACATTCTTCACTGTCTGTTGTTTACCTACC3151              GTAGCTTTTTACCGTTCACTTCCCCTTCCAACTATGTCCAGATGTGCAGGCTCCTCCTCT3211              CTGGACTTTCTCCAAAGGCACTGACCCTCGGCCTCTACTTTGTCCCCTCACCTCCACCCC3271              CTCCTGTCACCGGCCTTGTGACATTCACTCAGAGAAGACCACA CCAAGGAGGGGCCGCGG3331             CTGGCCCAGGAGAGAACACGGGGAGGTTTGTTTGTGTGAAAGGAAAGTAGTCCAGGCTGT3391              CCCTGAAACTGAGTCTGTGGACACTGTGGAAAGCTTTGAACAATTGTGTTTTCGTCACAG3451              GAGTCTTTGTAATGCTTGTA CAGTTGATGTCGATGCTCACTGCTTCTGCTTTTTCTTTCT3511             TTTTATTTTAAAAAATCTGAAGGTTCTGGTAACCTGTGGTGTATTTTTATTTTCCTGTGA3571              CTGTTTTTGTTTTGTTTTTTTCCTTTTTCCTCCCCTTTAGCCCTATTCATGTCTCTACCC3631              ACTATGCACAGATTAAACTTCACCTACAAACTCCTTAATATGATCTGTGGAGAATGTACA3691              CAGTTTAAACACATCAATAAATACTTTAACTTCC3725                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 763 amino acids                                                    (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      MetAlaAlaThrGlyThrAlaAlaAlaAlaAlaThrGlyArgLeuLeu                              151015                                                                        LeuLeuLeu LeuValGlyLeuThrAlaProAlaLeuAlaLeuAlaGly                             202530                                                                        TyrIleGluAlaLeuAlaAlaAsnAlaGlyThrGlyPheAlaValAla                              35 4045                                                                       GluProGlnIleAlaMetPheCysGlyLysLeuAsnMetHisValAsn                              505560                                                                        IleGlnThrGlyLysTrpGluProAspProThrGlyThrLy sSerCys                             65707580                                                                      PheGluThrLysGluGluValLeuGlnTyrCysGlnGluMetTyrPro                              85909 5                                                                       GluLeuGlnIleThrAsnValMetGluAlaAsnGlnArgValSerIle                              100105110                                                                     AspAsnTrpCysArgArgAspLysLysGlnCysLysSerArgPheVal                               115120125                                                                    ThrProPheLysCysLeuValGlyGluPheValSerAspValLeuLeu                              130135140                                                                     ValProGluLysCysGlnPhePheHisLys GluArgMetGluValCys                             145150155160                                                                  GluAsnHisGlnHisTrpHisThrValValLysGluAlaCysLeuThr                              165170 175                                                                    GlnGlyMetThrLeuTyrSerTyrGlyMetLeuLeuProCysGlyVal                              180185190                                                                     AspGlnPheHisGlyThrGluTyrValCysCysProGlnThrLy sIle                             195200205                                                                     IleGlySerValSerLysGluGluGluGluGluAspGluGluGluGlu                              210215220                                                                     GluGluGluAspGluGlu GluAspTyrAspValTyrLysSerGluPhe                             225230235240                                                                  ProThrGluAlaAspLeuGluAspPheThrGluAlaAlaValAspGlu                              245 250255                                                                    AspAspGluAspGluGluGluGlyGluGluValValGluAspArgAsp                              260265270                                                                     TyrTyrTyrAspThrPheLysGlyAspAspTyr AsnGluGluAsnPro                             275280285                                                                     ThrGluProGlySerAspGlyThrMetSerAspLysGluIleThrHis                              290295300                                                                     AspValL ysAlaValCysSerGlnGluAlaMetThrGlyProCysArg                             305310315320                                                                  AlaValMetProArgTrpTyrPheAspLeuSerLysGlyLysCysVal                               325330335                                                                    ArgPheIleTyrGlyGlyCysGlyGlyAsnArgAsnAsnPheGluSer                              340345350                                                                     GluAspTyrCysMetAlaVal CysLysAlaMetIleProProThrPro                             355360365                                                                     LeuProThrAsnAspValAspValTyrPheGluThrSerAlaAspAsp                              370375 380                                                                    AsnGluHisAlaArgPheGlnLysAlaLysGluGlnLeuGluIleArg                              385390395400                                                                  HisArgAsnArgMetAspArgValLysLysGluTrpGluGluAlaGlu                              405410415                                                                     LeuGlnAlaLysAsnLeuProLysAlaGluArgGlnThrLeuIleGln                              420425430                                                                     HisPheGlnA laMetValLysAlaLeuGluLysGluAlaAlaSerGlu                             435440445                                                                     LysGlnGlnLeuValGluThrHisLeuAlaArgValGluAlaMetLeu                              450455 460                                                                    AsnAspArgArgArgMetAlaLeuGluAsnTyrLeuAlaAlaLeuGln                              465470475480                                                                  SerAspProProArgProHisArgIleLeuGlnAla LeuArgArgTyr                             485490495                                                                     ValArgAlaGluAsnLysAspArgLeuHisThrIleArgHisTyrGln                              500505510                                                                     HisValLeuAlaValAspProGluLysAlaAlaGlnMetLysSerGln                              515520525                                                                     ValMetThrHisLeuHisValIleGluGluArgArgAsnGlnIleLeu                              530 535540                                                                    SerLeuLeuTyrLysValProTyrValAlaGlnGluIleGlnGluGlu                              545550555560                                                                  IleAspGluLeuLeuGlnGluGlnA rgAlaAspMetAspGlnPheThr                             565570575                                                                     AlaSerIleSerGluThrProValAspValArgValSerSerGluGlu                              580585 590                                                                    SerGluGluIleProProPheHisProPheHisProPheProAlaLeu                              595600605                                                                     ProGluAsnGluAspThrGlnProGluLeuTyrHisProMetLysLys                               610615620                                                                    GlySerGlyValGlyGluGlnAspGlyGlyLeuIleGlyAlaGluGlu                              625630635640                                                                  LysValIleAsnSe rLysAsnLysValAspGluAsnMetValIleAsp                             645650655                                                                     GluThrLeuAspValLysGluMetIlePheAsnAlaGluArgValGly                              660 665670                                                                    GlyLeuGluGluGluArgGluSerValGlyProLeuArgGluAspPhe                              675680685                                                                     SerLeuSerSerSerAlaLeuIleGlyLeuLeuValI leAlaValAla                             690695700                                                                     IleAlaThrValIleValIleSerLeuValMetLeuArgLysArgGln                              705710715720                                                                  Tyr GlyThrIleSerHisGlyIleValGluValAspProMetLeuThr                             725730735                                                                     ProGluGluArgHisLeuAsnLysMetGlnAsnHisGlyTyrGluAsn                               740745750                                                                    ProThrTyrLysTyrLeuGluGlnMetGlnIle                                             755760                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Primer 1                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GCTGAGAGATTGGAGAAGAGAGAGGTGTGCTCTGAAC37                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Primer 2                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CTGCTATCTAGATTAGGCGCTGCCACACACGGC33                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 168 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: APP                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..168                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GAGG TGTGCTCTGAACAAGCCGAGACGGGGCCGTGCCGAGCAATGATC48                           GluValCysSerGluGlnAlaGluThrGlyProCysArgAlaMetIle                              151015                                                                        TCC CGCTGGTACTTTGATGTGACTGAAGGGAAGTGTGCCCCATTCTTT96                           SerArgTrpTyrPheAspValThrGluGlyLysCysAlaProPhePhe                              202530                                                                        TACGGC GGATGTGGCGGCAACCGGAACAACTTTGACACAGAAGAGTAC144                          TyrGlyGlyCysGlyGlyAsnArgAsnAsnPheAspThrGluGluTyr                              354045                                                                        TGCATGGCCGT GTGTGGCAGCGCC168                                                  CysMetAlaValCysGlySerAla                                                      5055                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GluValCysSerGluGlnAlaGluThrGlyProCysArgAlaMetIle                              151015                                                                        SerArgTrpTyrPheAspValThrGluGlyL ysCysAlaProPhePhe                             202530                                                                        TyrGlyGlyCysGlyGlyAsnArgAsnAsnPheAspThrGluGluTyr                              354045                                                                         CysMetAlaValCysGlySerAla                                                     5055                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 1..4                                                           (D) OTHER INFORMATION: /label=Ala-1                                           /note="Amino-terminal alanine residue is capped                               with a methoxysuccinyl gr..."                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /label=Val-4                                           /note="Carboxyl-terminal valine residue is capped                             with p- nitroznilide"                                                         (x i) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                     AlaAlaProVal                                                                  (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1..4                                                             (D) OTHER INFORMATION: /label=ALA-1                                          /note="Amino terminal alanine residue is capped                               with a succinyl group"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /label=Phe-4                                           /note="Carboxyl-terminal phenylalanine resudue is                             capped with p-nitroanil..."                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AlaAla ProPhe                                                                 1                                                                         

We claim:
 1. An isolated human Kunitz-type inhibitor comprising theamino acid sequence of SEQ ID NO:2 from alanine, amino acid 56 toalanine, amino acid number 110; the amino acid sequence of SEQ ID NO:2from aspartic acid, amino acid number 53 to alanine, amino acid number110; or the amino acid sequence of SEQ ID NO:2 from valine, amino acidnumber 57 to alanine, amino acid number 110 and which further contains aglutamic acid residue on the amino terminus.
 2. A pharmaceuticalcomposition which comprises a human Kunitz-type inhibitor of claim 1 incombination with a pharmaceutically acceptable carrier or vehicle.
 3. Anisolated amyloid protein precursor homologue comprising the amino acidsequence of SEQ ID NO:13 from methionine, amino acid number 1, toisoleucine, amino acid number 763.